CRITERION™ CYTOPHAGA AGAR BASE

Cat. no. C7730 CRITERION™ Cytophaga Agar Base 32.8gm
Cat. no. C7731 CRITERION™ Cytophaga Agar Base 500gm
Cat. no. C7732 CRITERION™ Cytophaga Agar Base 2kg
Cat. no. C7733 CRITERION™ Cytophaga Agar Base 10kg
Cat. no. C7734 CRITERION™ Cytophaga Agar Base 50kg

INTENDED USE

Hardy Diagnostics CRITERION™ Cytophaga Agar Base is recommended for the cultivation and maintenance of Cytophaga species and Flavobacterium species.

This dehydrated culture medium is a raw material intended to be used in the making of prepared media products, which will require further processing, additional ingredients, or supplements.

SUMMARY

Cold-water disease, bacterial gill disease (BGD), peduncle disease, columnaris, and other fish diseases commonly caused by bacterium in the Cytophaga-Flavobacterium-Bacteroides (CFB) group are usually associated with abrupt changes in environmental conditions such as extremes in water temperature, low dissolved oxygen concentration, overcrowding, poor diet, or improper handling. Many of these bacteria are ubiquitous in most, if not all, aquaculture environments and probably play a role in the turnover of organic matter, but some species may become opportunistic pathogens when fish are stressed.(1) Infection is most commonly associated with fry or fingerling populations and lesions may appear in various locations on different species of fish or may involve systemic infections with no visible signs of disease. Outbreaks can be highly contagious and, under the appropriate conditions, infection can spread rapidly, resulting in catastrophic losses and economic hardship in conservation hatcheries and the commercial aquaculture industry in a matter of days. Moreover, some bacteria can spread horizontally, from fish to fish, or vertically, from parent to offspring, and result in secondary infections or other diseases that can quickly overwhelm a population.

Researchers have devised many suitable low nutrient media in order to recover these etiological agents of disease, but Cytophaga Media has received the greatest acceptance.(2) Cytophaga Media is a general, non-selective medium that supports the growth of a wide variety of microorganisms; thus, knowledge of cell and colony morphology is essential.(3) Species in the Cytophaga-Flavobacterium-Bacteroides (CFB) group generally exhibit gram-negative, rod shaped cells of varying length and width, produce a yellow, orange, or brick red pigment and may swarm by gliding or produce a biofilm. The low nutrient concentration of this medium mimics the natural habitat conditions of these opportunistic pathogens and helps promote their isolation by minimizing swarming.

Hardy Diagnostics CRITERION™ Cytophaga Agar Base contains casein peptone, yeast extract and beef extract which provide essential amino acids and other nitrogenous compounds. Sodium acetate is the sodium salt of acetic acid and is used as a buffering agent. Agar is the solidifying agent. The media can be enriched with ingredients (e.g. serum, gelatin and starch) for fastidious microorganisms or supplemented with selective agents to make Selective Cytophaga Agar (SCA) for greater isolation of Cytophaga species and Flavobacterium columnare from mixed cultures. It can also be prepared with 70% seawater for the cultivation and maintenance of marine microorganisms like Flexibacter maritimus, or other marine bacteria recognized as global contributors to diseases in salmon.(1,7)

FORMULA*

Gram weight per liter: 16.4gm/L
Casein Peptone 0.5gm
Yeast Extract 0.5gm
Beef Extract 0.2gm
Sodium Acetate 0.2gm
Agar 15.0gm

Final pH 7.0 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Store the sealed bottle(s) containing dehydrated culture medium at 2-30ºC. Dehydrated culture medium is very hygroscopic. Keep lid tightly sealed. Protect dehydrated culture media from moisture and light. The dehydrated culture media should be discarded if it is not free-flowing or if the color has changed from its original beige

Store the prepared culture media at 2-8ºC.

PRECAUTIONS

METHOD OF PREPARATION FOR DEHYDRATED CULTURE MEDIA

1. Suspend 16.4gm of the dehydrated culture media in 1 liter of distilled or deionized water. Stir to mix thoroughly.

Note: It is recommended that at least 70% sterile seawater and inorganic salts such as KCl and NaCl be used for the optimum cultivation of marine microorganisms.

2. Check pH and adjust if necessary.

3. Sterilize in the autoclave at 121ºC. for 15 minutes.

4. Cool to 45-50ºC.

Note: Neomycin (5mg/L) and polymyxin B (200 units/mL) can be added, if desired, to make Selective Cytophaga Agar (SCA).

5. Aseptically dispense desired volume into appropriate sterile containers.

PROCEDURE AND INTERPRETATION OF RESULTS

For information on procedures and interpretation of results, consult listed references.

LIMITATIONS

Organisms belonging to the Cytophaga-Flavobacterium-Bacteroides (CFB) group are organotrophs and may be either aerobic, microaerophilic, capnophilic (CO2 requiring), or facultatively anaerobic.(1) Appropriate incubation procedures should be followed for best results.

F. branchiophilum are fastidious and enrichment of the medium with serum, gelatin or starch is recommended.(13)

Columnaris bacteria are best grown on selective media such as Selective Cytophaga Agar containing neomycin (5mg/L) and polymyxin B (200 units/mL), whereas most other fish pathogens and aquatic bacteria are inhibited.(4)

Flexibacter maritimus will only grow on media prepared with at least 50% seawater. In addition, the bacterium requires the use of two inorganic salts (KCl and NaCl) for optimum growth.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as autoclaves, incinerators, antibiotics, sea water, inorganic salts, serum, gelatin, starch and incubators, etc., are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Cytophaga spp.
ATCC ® 53485
A 24-48hr 15-30°C Aerobic Growth

User Quality Control

Physical Appearance

CRITERION™ Cytophaga Agar Base powder should appear homogeneous, free-flowing, and beige in color. The prepared media should appear clear, and light beige in color.

REFERENCES

1. Dworkin, Martin. 2006. The Prokaryotes, 3rd ed. Vol. 7. Springer, New York, NY.

2. Austin, Brian. 1999. Bacterial Fish Pathogens: Disease of Farmed and Wild Fish. Springer. New York, NY.

3. Couch, J.A. and J.W. Fournie. 1993. Pathobiology of Marine and Estuarine Organisms. Center for Marine and Estuarine Disease. Gulf Breeze, FL.

4. Durborow, R.M., R.L. Thune, J.P. Hawke and A.C. Camus. 1998. Columnaris Disease, A bacterial Infection Caused by Flavobacterium columnare. SRAC Publication No. 479.

5. LaFrentz, B.R and K.D. Cain. 2004. Bacterial Coldwater Disease, An Extension Bulletin for the Western Regional Aquaculture Center (WRAC). WRAC Publications. University of Idaho. Moscow, ID.

6. Cipriano, R.C. and R.A. Holt. 2005. Flavobacterium psychrophuilum, cause of Bacterial Cold-Water Disease and Rainbow Trout Fry Syndrome. Fish Disease Leaflet No. 86. U.S. Dept. of the Interior, U.S. Geol. Survey, Natl. Fish Health Research Lab. Kearneysville, WV.

7. Santos, Y., F. Pazos and J.L. Barja. 1999. Flexibacter maritimus, causal agent of flexibacteriosis in marine fish. Leaflet No. 55. International Council for the Exploration of the Sea. Copenhagen, Denmark.

8. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

9. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

10. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA.
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm.

11. Pilarski, F., A.J. Rossini and P.S. Ceccarelli. 2008. Isolation and characterization of Flavobacterium columnare (Bernardet et al. 2002) from four tropical fish species in Brazil. Braz. J. Biol.; 68(2):409-414.

12. Bernardet, J.F., et al. 1996. Cutting a Gordian Knot: Emended Classification and Description of the Genus Flavobacterium, Emended Description of the Family Flavobacteriaceae, and Proposal of Flavobacterium hydatis nom. nov. (Basonym, Cytophaga aquatilis Strohl and Tait 1978). Intl. J. of Sys. Bact.; 46:1.

13. Holt, J.G. et al. 1994. Bergey's Manual of Determinative Bacteriology, 9th ed. Williams and Wilkins. Baltimore, MD.


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051616gr